Dual DNA rulers reveal an 'mRNA looping' intermediate state during ribosome translocation.
ABSTRACT: The precise 3-nucleotide movement of mRNA is critical for translation fidelity. One mRNA translocation error propagates to all of the following codons, which is detrimental to the cell. However, none of the current methods can reveal the mRNA dynamics near the ribosome entry site, which limits the understanding of this important issue. We have developed an assay of dual DNA rulers that provides such capability. By uniquely probing both the 3'- and 5'-ends of mRNA, we observed an antibiotic-trapped intermediate state that is consistent with a ribosomal conformation containing mRNA asymmetric partial displacements at its entry and exit sites. Based on the available ribosome structures and computational simulations, we proposed a 'looped' mRNA conformation, which suggested a stepwise 'inchworm' mechanism for ribosomal translocation. The same 'looped' intermediate state identified with the dual rulers persists with a '-1' frameshifting motif, indicating that the branching point of normal and frameshifting translocations occurs at a later stage of translocation.
Project description:Programmed ribosomal frameshifting produces alternative proteins from a single transcript. -1 frameshifting occurs on Escherichia coli's dnaX mRNA containing a slippery sequence AAAAAAG and peripheral mRNA structural barriers. Here, we reveal hidden aspects of the frameshifting process, including its exact location on the mRNA and its timing within the translation cycle. Mass spectrometry of translated products shows that ribosomes enter the -1 frame from not one specific codon but various codons along the slippery sequence and slip by not just -1 but also -4 or +2 nucleotides. Single-ribosome translation trajectories detect distinctive codon-scale fluctuations in ribosome-mRNA displacement across the slippery sequence, representing multiple ribosomal translocation attempts during frameshifting. Flanking mRNA structural barriers mechanically stimulate the ribosome to undergo back-and-forth translocation excursions, broadly exploring reading frames. Both experiments reveal aborted translation around mutant slippery sequences, indicating that subsequent fidelity checks on newly adopted codon position base pairings lead to either resumed translation or early termination.
Project description:Internal ribosome entry sites (IRESs) mediate cap-independent translation of viral mRNAs. Using electron cryo-microscopy of a single specimen, we present five ribosome structures formed with the Taura syndrome virus IRES and translocase eEF2•GTP bound with sordarin. The structures suggest a trajectory of IRES translocation, required for translation initiation, and provide an unprecedented view of eEF2 dynamics. The IRES rearranges from extended to bent to extended conformations. This inchworm-like movement is coupled with ribosomal inter-subunit rotation and 40S head swivel. eEF2, attached to the 60S subunit, slides along the rotating 40S subunit to enter the A site. Its diphthamide-bearing tip at domain IV separates the tRNA-mRNA-like pseudoknot I (PKI) of the IRES from the decoding center. This unlocks 40S domains, facilitating head swivel and biasing IRES translocation via hitherto-elusive intermediates with PKI captured between the A and P sites. The structures suggest missing links in our understanding of tRNA translocation.
Project description:Programmed -1 ribosomal frameshifting (-1PRF) is an mRNA recoding event utilized by cells to enhance the information content of the genome and to regulate gene expression. The mechanism of -1PRF and its timing during translation elongation are unclear. Here, we identified the steps that govern -1PRF by following the stepwise movement of the ribosome through the frameshifting site of a model mRNA derived from the IBV 1a/1b gene in a reconstituted in vitro translation system from Escherichia coli. Frameshifting occurs at a late stage of translocation when the two tRNAs are bound to adjacent slippery sequence codons of the mRNA. The downstream pseudoknot in the mRNA impairs the closing movement of the 30S subunit head, the dissociation of EF-G, and the release of tRNA from the ribosome. The slippage of the ribosome into the -1 frame accelerates the completion of translocation, thereby further favoring translation in the new reading frame.
Project description:Ribosomal frameshifting occurs when a ribosome slips a few nucleotides on an mRNA and generates a new sequence of amino acids. Programmed -1 ribosomal frameshifting (-1PRF) is used in various systems to express two or more proteins from a single mRNA at precisely regulated levels. We used single-molecule fluorescence resonance energy transfer (smFRET) to study the dynamics of -1PRF in the Escherichia coli dnaX gene. The frameshifting mRNA (FSmRNA) contained the frameshifting signals: a Shine-Dalgarno sequence, a slippery sequence, and a downstream stem loop. The dynamics of ribosomal complexes translating through the slippery sequence were characterized using smFRET between the Cy3-labeled L1 stalk of the large ribosomal subunit and a Cy5-labeled tRNA(Lys) in the ribosomal peptidyl-tRNA-binding (P) site. We observed significantly slower elongation factor G (EF-G)-catalyzed translocation through the slippery sequence of FSmRNA in comparison with an mRNA lacking the stem loop, ?SL. Furthermore, the P-site tRNA/L1 stalk of FSmRNA-programmed pretranslocation (PRE) ribosomal complexes exhibited multiple fluctuations between the classical/open and hybrid/closed states, respectively, in the presence of EF-G before translocation, in contrast with ?SL-programmed PRE complexes, which sampled the hybrid/closed state approximately once before undergoing translocation. Quantitative analysis showed that the stimulatory stem loop destabilizes the hybrid state and elevates the energy barriers corresponding to subsequent substeps of translocation. The shift of the FSmRNA-programmed PRE complex equilibrium toward the classical/open state and toward states that favor EF-G dissociation apparently allows the PRE complex to explore alternative translocation pathways such as -1PRF.
Project description:Maintenance of translational reading frame ensures the fidelity of information transfer during protein synthesis. Yet, programmed ribosomal frameshifting sequences within the coding region promote a high rate of reading frame change at predetermined sites thus enriching genomic information density. Frameshifting is typically stimulated by the presence of 3' messenger RNA (mRNA) structures, but how these mRNA structures enhance -1 frameshifting remains debatable. Here, we apply single-molecule and ensemble approaches to formulate a mechanistic model of ribosomal -1 frameshifting. Our model suggests that the ribosome is intrinsically susceptible to frameshift before its translocation and this transient state is prolonged by the presence of a precisely positioned downstream mRNA structure. We challenged this model using temperature variation in vivo, which followed the prediction made based on in vitro results. Our results provide a quantitative framework for analyzing other frameshifting enhancers and a potential approach to control gene expression dynamically using programmed frameshifting.
Project description:The triplet-based genetic code requires that translating ribosomes maintain the reading frame of a messenger RNA faithfully to ensure correct protein synthesis. However, in programmed -1 ribosomal frameshifting, a specific subversion of frame maintenance takes place, wherein the ribosome is forced to shift one nucleotide backwards into an overlapping reading frame and to translate an entirely new sequence of amino acids. This process is indispensable in the replication of numerous viral pathogens, including HIV and the coronavirus associated with severe acute respiratory syndrome, and is also exploited in the expression of several cellular genes. Frameshifting is promoted by an mRNA signal composed of two essential elements: a heptanucleotide 'slippery' sequence and an adjacent mRNA secondary structure, most often an mRNA pseudoknot. How these components operate together to manipulate the ribosome is unknown. Here we describe the observation of a ribosome-mRNA pseudoknot complex that is stalled in the process of -1 frameshifting. Cryoelectron microscopic imaging of purified mammalian 80S ribosomes from rabbit reticulocytes paused at a coronavirus pseudoknot reveals an intermediate of the frameshifting process. From this it can be seen how the pseudoknot interacts with the ribosome to block the mRNA entrance channel, compromising the translocation process and leading to a spring-like deformation of the P-site transfer RNA. In addition, we identify movements of the likely eukaryotic ribosomal helicase and confirm a direct interaction between the translocase eEF2 and the P-site tRNA. Together, the structural changes provide a mechanical explanation of how the pseudoknot manipulates the ribosome into a different reading frame.
Project description:Reading frame maintenance is critical for accurate translation. We show that the conserved eukaryotic/archaeal protein Mbf1 acts with ribosomal proteins Rps3/uS3 and eukaryotic Asc1/RACK1 to prevent frameshifting at inhibitory CGA-CGA codon pairs in the yeast Saccharomyces cerevisiae. Mutations in RPS3 that allow frameshifting implicate eukaryotic conserved residues near the mRNA entry site. Mbf1 and Rps3 cooperate to maintain the reading frame of stalled ribosomes, while Asc1 also mediates distinct events that result in recruitment of the ribosome quality control complex and mRNA decay. Frameshifting occurs through a +1 shift with a CGA codon in the P site and involves competition between codons entering the A site, implying that the wobble interaction of the P site codon destabilizes translation elongation. Thus, eukaryotes have evolved unique mechanisms involving both a universally conserved ribosome component and two eukaryotic-specific proteins to maintain the reading frame at ribosome stalls.
Project description:Downstream stable mRNA secondary structures can stall elongating ribosomes by impeding the concerted movements of tRNAs and mRNA on the ribosome during translocation. The addition of a downstream mRNA structure, such as a stem-loop or a pseudoknot, is essential to induce -1 programmed ribosomal frameshifting (-1 PRF). Interestingly, previous studies revealed that -1 PRF efficiencies correlate with conformational plasticity of pseudoknots, defined as their propensity to form incompletely folded structures, rather than with the mechanical properties of pseudoknots. To elucidate the detailed molecular mechanisms of translocation and -1 PRF, we applied several smFRET assays to systematically examine how translocation rates and conformational dynamics of ribosomes were affected by different pseudoknots. Our results show that initial pseudoknot-unwinding significantly inhibits late-stage translocation and modulates conformational dynamics of ribosomal post-translocation complexes. The effects of pseudoknots on the structural dynamics of ribosomes strongly correlate with their abilities to induce -1 PRF. Our results lead us to propose a kinetic scheme for translocation which includes an initial power-stroke step and a following thermal-ratcheting step. This scheme provides mechanistic insights on how selective modulation of late-stage translocation by pseudoknots affects -1 PRF. Overall our findings advance current understanding of translocation and ribosome-induced mRNA structure unwinding.
Project description:The ribosome translates the genetic information encoded in messenger RNA into protein. Folded structures in the coding region of an mRNA represent a kinetic barrier that lowers the peptide elongation rate, as the ribosome must disrupt structures it encounters in the mRNA at its entry site to allow translocation to the next codon. Such structures are exploited by the cell to create diverse strategies for translation regulation, such as programmed frameshifting, the modulation of protein expression levels, ribosome localization and co-translational protein folding. Although strand separation activity is inherent to the ribosome, requiring no exogenous helicases, its mechanism is still unknown. Here, using a single-molecule optical tweezers assay on mRNA hairpins, we find that the translation rate of identical codons at the decoding centre is greatly influenced by the GC content of folded structures at the mRNA entry site. Furthermore, force applied to the ends of the hairpin to favour its unfolding significantly speeds translation. Quantitative analysis of the force dependence of its helicase activity reveals that the ribosome, unlike previously studied helicases, uses two distinct active mechanisms to unwind mRNA structure: it destabilizes the helical junction at the mRNA entry site by biasing its thermal fluctuations towards the open state, increasing the probability of the ribosome translocating unhindered; and it mechanically pulls apart the mRNA single strands of the closed junction during the conformational changes that accompany ribosome translocation. The second of these mechanisms ensures a minimal basal rate of translation in the cell; specialized, mechanically stable structures are required to stall the ribosome temporarily. Our results establish a quantitative mechanical basis for understanding the mechanism of regulation of the elongation rate of translation by structured mRNAs.
Project description:It has been characterized that the programmed ribosomal -1 frameshifting often occurs at the slippery sequence on the presence of a downstream mRNA pseudoknot. In some prokaryotic cases such as the dnaX gene of Escherichia coli, an additional stimulatory signal-an upstream, internal Shine-Dalgarno (SD) sequence-is also necessary to stimulate the efficient -1 frameshifting. However, the molecular and physical mechanism of the -1 frameshifting is poorly understood. Here, we propose a model of the pathway of the -1 translational frameshifting during ribosome translation of the dnaX -1 frameshift mRNA. With the model, the single-molecule fluorescence data (Chen et al. (2014) ) on the dynamics of the shunt either to long pausing or to normal translation, the tRNA transit and sampling dynamics in the long-paused rotated state, the EF-G sampling dynamics, the mean rotated-state lifetimes, etc., are explained quantitatively. Moreover, the model is also consistent with the experimental data (Yan et al. (2015) ) on translocation excursions and broad branching of frameshifting pathways. In addition, we present some predicted results, which can be easily tested by future optical trapping experiments.